Table 1.

SMase natural substrate assay protocol (pH 6.5)

Step	Parameter	Value		Description
		384-well plate	1,536-well plate
1	Enzyme solution	20 μl/well	2 μl/well	153 nM SMase in buffer
2	Compound	0.2 μl/well	0.02 μl/well	In DMSO solution
3	Substrate solution	20 μl/well	2 μl/well	100 μM sphingomyelin in buffer
4	Incubation	5 h	5 h	37 °C
5	Detection mixture	20 μl/well	2 μl/well	From the assay kit
6	Amplite-Red dye	10 μl/well	1 μl/well	From the assay kit
7	Incubation	120 min	120 min	Room temperature
8	Plate reading	Ex0540 nm Em0590 nm	Ex0540 nm Em0590 nm	Fluorescence intensity